Recruitment of Subjects
In this case–control study, a total of 178 subjects were recruited of which 105 were drug naïve PCOS women and 73 were age-matched healthy controls. PCOS cases were recruited from the patients attending OPD of Department of Endocrinology, Sher-e-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, Jammu and Kashmir, India and Department of Obstetrics and Gynaecology, Govt. Medical College, Srinagar, Jammu and Kashmir, India for PCOS related complications from October 2017 to March 2020. The inclusion criteria for PCOS patients was based on the Rotterdam criteria 10. All those subjects who presented with PCOS mimicking disorders including thyroid dysfunction , hyperprolactinemia , congenital adrenal hyperplasia, androgen secreting tumours and Cushing’s syndrome were excluded from the study. Controls recruited for the study were age matched healthy women with regular cycles, no signs of hyperandrogenism clinical/ biochemical, with no history of autoimmune or endocrine disorders. All of the subjects were ethnic Kashmiris living in Kashmir province
This study was approved by the ethical committee Government Medical College Srinagar under ethical approval no. 94/ETH/GMC/ICMR. All subjects were recruited after written informed consent was obtained from them.
Anthropometric and clinical evaluation
All the subjects underwent general anthropometric measurements that include height, weight, waist, hip, waist-hip ratio (WHR), body mass index (BMI) and FG score. A detailed history of clinical symptoms like menstrual cycles, hirsutism, acne, alopecia, and acanthosis nigricans was also taken from all study subjects. Height was measured using a height measuring scale in standing position without shoes and weight was measured using a weighing scale with light clothing. For measurement of waist circumference, minimum value between the iliac crest and the lateral costal margin was determined in a standing position, and hip circumference was measured as the maximum value over the buttocks. WHR was calculated by dividing waist circumference by the hip circumference. BMI was calculated as weight(kg)/Height(m²). Hirsutism was measured by Ferriman-Gallwey scoring system and a score of greater than 8 was taken as significant. All the PCOS women underwent transabdominal ultrasonography (USG) to measure the number of small peripheral cysts and/or increased ovarian volume.
Hormonal and Biochemical assessment
For hormonal and biochemical assessment, blood sample was collected in clot activator vials from all the participants after an overnight fast on 2nd – 3rd day of their menstrual cycle. The hormonal profile included Testosterone, luteinizing hormone, Follicle stimulating hormone. Thyroid stimulating hormone to exclude thyroid disorders, Prolactin to exclude hyperprolactinemia were estimated by Radioimmunoassay (RIA) using RIA kits (Immunotech S.R.O, Prague, Czech Republic) on Beckman coulter UniCelDxl 800 (Access Immunoassay system). Enzyme-linked immunosorbent assays were used to measure Sex Hormone-Binding Globulin (SHBG), androstenedione, dehydroepiandrosterone sulfate (DHEAS), and fasting insulin using Calbiotech, CA USA and DGR Instruments GmbH Marburg ELISA kits using SkanIt RE 4.0 software on Thermo Scientific Multiskan FC ELISA reader. The biochemical parameters done included oral glucose tolerance test, cholesterol, triglyceride, High Density Lipoprotein cholesterol levels, Low-Density Lipoprotein cholesterol levels, Urea, Creatinine, SGOT, SGPT. All the biochemical parameters were measured on semi-automatic analyzer (ERBA Chemtouch 7, Biochemistry Analyzer, Wiesbaden, Germany) using ERBA bioassay diagnostic kits.
The free androgen index (FAI) was derived using the formula:
Insulin resistance was calculated
Quantitative Insulin Sensitivity Check Index (QUICKI) was calculated by formula:
Peripheral blood mononuclear cell isolation
For isolation of peripheral blood mononuclear cell 2ml blood sample in Na-EDTA vials was obtained from all the subjects. Blood samples were diluted by addition of equal volume of phosphate buffer saline (PBS) followed by density gradient centrifugation using Ficoll-Paque plus (GE Healthcare Bio-Sciences Sweden). After density gradient centrifugation Peripheral blood mononuclear cell (PBMNCs) were isolated and washed twice with PBS to remove any contamination of Ficoll and plasma. The cells thus obtained were stored at -80°C for further processing.
RNA isolation and cDNA Synthesis
Total RNA was isolated from blood leukocytes by TRIzol method 11. All of the RNA samples were subjected to DNase treatment using Sigma Aldrich DNase treatment kit according to manufacturer’s protocol in order to eliminate any traces of genomic DNA. The RNA thus obtained was checked for integrity on 2% agarose gel. Qualitative and quantitative analysis of all the RNA samples was done on NanoDrop (Thermo Scientific). All RNA samples with an absorbance A260/280 ratio of 1.9–2.0 were subsequently used for cDNA synthesis by RT-PCR. 1.5mg of total RNA was reverse transcribed into cDNA using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit as per manufacturer’s protocol in applied biosystems thermal cycler.
Quantitative real-time polymerase chain reaction
Expression levels of lncRNA CTBP1-AS were measured by Quantitative real-time polymerase chain reaction (qRT-PCR) using 10ml KAPA SYBR® FAST SYBR green , 0.3ml of forward and 0.3ml of reverse primers and cDNA less than 100ng (1ml) in a 20ml reaction following manufacturer’s protocol. qRT-PCR was performed in Roche LightCycler® 480 Instrument II 96 well plate having following reaction protocol pre-incubation at 95°C for 5mins, amplification for 40 cycles at 95°C for 20 sec, 62°C for 15 sec and 72°C for 15 sec. Amplification was followed by melting curve analysis with following conditions 95°C for 5sec, 60°C for 1min and 95°C continuous. The amplified product was subsequently run on 2% agarose gel to confirm product size. The relative expression of CTBP1-AS was estimated by Livak method and Beta-Actin was used as reference gene 12. Each reaction of qRT-PCR was performed in triplicates.
The following primers were used to quantify the lncRNA CTBP1-AS levels: 5´-TTGATGAGGTGGTGGTTGTG -3´ (forward) and 5´ - GACCCTTACTTGTCGGATGG -3´ (reverse), β-Actin RNA was quantified as a control to normalize differences in total RNA levels using the following primers 5´ - ATCGGAACGGTGAAGGTGACA -3´ (forward) and 5´- ATGGCAAGGGACTTCCTGTAAC -3´ (reverse).
All baseline quantitative variables were expressed as mean ± SD. The anthropometric, hormonal and biochemical parametric variables between PCOS and controls and were compared by unpaired student t-test. Chi square test was used to study association of CTBP1-AS expression and various non-parametric variables. The relation between different metabolic and endocrine markers with CTBP1-AS was evaluated by Pearson or Spearman rank correlation coefficient. The expression of CTBP1-AS was divided into binary groups in both PCOS cases and in healthy controls based on limits derived from the healthy control group as follows <1.81 for the low expression and >1.81 for high expression. Receiver operator characteristic (ROC) and area under curve (AUC) was calculated using Sigma Plot 10. The statistical analysis was done using statistical computing tool vassarstats (http://vassarstats.net/). P- value of <0.05 was considered as statistically significant.